The mammalian brain is composed of billions of neurons. Each of these highly specialized cells is estimated to make hundreds, if not thousands, of synaptic connections to other neurons. The staggering complexity of this network parallels the remarkable intellectual and creative abilities of the human mind. Only a fraction of these circuits have been defined at the level of individual cells, however. And it seems unlikely that we can even begin to understand the origins of these complex behaviors until a much more detailed wiring diagram of the vertebrate brain is known. The purpose of this proposal is to develop microscopic methods that can reveal synapses between specific neurons. We have chosen to test the reliability of these approaches in a model organism with a simple, well defined nervous system. In the nematode C. elegans the nervous system is composed of precisely 302 neurons. The morphology and synaptic connectivity of each of these neurons has been defined by serial section electron microscopy. Thus, we have a complete circuit diagram for the entire C. elegans nervous system. Our goal is to test the feasibility of observing these neurons and their specific synapses in intact animals in the light microscope. Because the methods that we will develop would obviate the need for EM reconstruction, our approaches should greatly facilitate efforts to identify specific mutations that perturb the wiring diagram of this simple nervous system and thereby lead to the cloning of the genes that control synaptic specificity. In addition, it is expected that the methods that we will develop can be utilized in the future to define the detailed neuroanatomy of other, more complex nervous systems. We will develop methods that can be utilized to observe specific subsets of these connections in the light microscope. Our strategy exploits the following tools: 1) green fluorescent protein (GFP) and epitope tags to label synapse-specific proteins and the neurons in which they are expressed; 2) cloned gene regulatory regions to drive expression of these marked proteins in specific neurons; 3) confocal and multiphoton excitation microscopy to resolve these labeled neuronal processes and their synaptic connections.